The detection of biologically important thiols has been the focus of much research. Different naturally-occurring thiols, which may have similar structures, may have quite different physiological properties. The physiological effects and correlations that have been observed for these thiols are a public health concern. Examples of low molecular weight thiols that have more-or-less similar structures, but that have disparate physiological properties, include cysteine (Cys), homocysteine (Hcy), glutathione (GSH), N-acetylcysteine, and penicillamine.
Glutathione is of particular interest to medical researchers. Glutathione levels are indicative of oxidative stress. Additionally, low glutathione levels may be linked, for example, to mitochondrial diseases, autism, and mercury poisoning.
Thiols are easily oxidized, and are typically colorless and non-fluorescent at visible wavelengths. Most reported methods for thiol detection have been based upon nonspecific redox chemistry, immunoassays, or upon derivatization with chromophores or fluorophores. Generic methods for detecting thiols do not readily distinguish among the structurally similar species. There is a substantial need for improved methods for detecting and quantifying biological thiols.
Methylviologen (MV2+) is a 4,4′-dipyridyl dication:
MV2+ has been used as an oxidant in an investigation of the equilibrium kinetics of both the reducing disulfide and the α-amino carbon-centered radicals derived from Hcy, Cys and GSH. Reducing radical formation was monitored via changes in the UV-Vis spectra of solutions containing the methylviologen radical cation that formed in the presence of the biological thiols. See R. Zhao et al., “Kinetics of one-electron oxidation of thiols and hydrogen abstraction by thiyl radicals from α-amino C—H bonds,” J. Am. Chem. Soc., vol. 116, pp. 12010-12015 (1994); and R. Zhao et al., “Significance of the intramolecular transformation of glutathione thiyl radicals to α-aminoalkyl radicals. Thermochemical and biological implications,” J. Chem. Soc., Perkins Trans., vol. 2, pp. 569-574 (1997) It was surmised that formation of the reducing α-aminoalkyl radical derived from Hcy should be particularly favorable, due to an intramolecular hydrogen abstraction mechanism involving a five-atom ring transition state (See FIG. 1A). By contrast, in the case of either Cys or GSH, H-atom abstraction to a reducing carbon-centered radical would proceed via less-favored four-membered ring (FIG. 1B) or nine-membered ring (not shown) transition state geometries, respectively. See FIGS. 1A and 1B, depicting the inferred proton abstraction leading to formation of the α-aminoalkyl radical from the thiyl radicals of Hcy and Cys, respectively. These references do not disclose any appreciable colorimetric selectivity among homocysteine, cysteine, and glutathione.
U.S. Publication 2008/0261315, which is incorporated herein by reference, discloses a method for selectively determining homocysteine with methylviologen. Heating a sample containing Hcy with a colorless solution of methylviologen for five minutes or longer at a temperature between about 25° C. and 1 10° C. and a pH between about 3.9 and about 9.5 produces a visible color change. Color formation can be monitored via the appearance of absorption peaks at 398 nm and 605 nm. In contrast, samples containing Cys or GSH remain colorless when heated with a solution of methylviologen under similar conditions.